There is ample evidence that cAMP is a second messenger involved in regulation of the function of the maturing spermatozoon. Both developing germ cells and spermatozoa are devoid of known receptors and Gs protein that in somatic cells serves to activate adenylate cyclase but express high levels of phosphodiesterases (PDEs). We therefore propose to test the hypothesis that PDEs and their regulation play a crucial role in the control of cAMP levels in the male gamete. Understanding the nature of the PDEs of the gamete is also important because pharmacological inhibition of these enzymes is an effective approach to manipulate cAMP levels for treatment of immotile spermatozoa. Our preliminary data demonstrate that two genes encoding a cAMP-specific PDE (cAMP-PDE) and a calmodulin- regulated PDE (CaM-PDE) are activated during spertmatogenesis and the PDE products of these genes are present in spermatozoa. The properties and distribution of the two PDEs will be followed by western analysis and immunocytochemistry. Since these forms are soluble in spermatids but recovered in the particulate fraction in spermatozoa, the protein modification causing this compartmentalization will be investigated. In view of the fact that acquisition of sperm motility in the epididymis is associated with an increase in cAMP and a reduction of the PDE activity, the mechanism leading to inhibition of the activity of the PDEs will be investigated. The PDE protein levels in spermatozoa from different portions of the epididymis will be quantitated by western analysis and related to the enzyme activity. The state of phosphorylation and the impact on activity of these enzymes from caput and cauda will be determined. Because caffeine, a non selective PDE inhibitor with low potency has been used to improve human sperm quality in infertile patients, a novel, pharmacological approach using form-selective inhibitors will be developed to manipulate human spermatozoon PDE activity. The PDE isoforms present in ejaculated human spermatozoa and their localization will be characterized using biochemical and immunological tools available. Novel classes of PDE inhibitors specific for the forms expressed in the spermatozoon will be used to study the effects of PDE inhibition on motility and acrosomal reaction. If the difference in PDE isoform distribution is found in human, isoform- selective inhibitors will be used to manipulate cAMP levels in the head or in the flagellum of the spermatozoon, thus selectively affecting motility or acrosomal reaction. In addition, we propose to establish and test a transgenic mouse model to study the role of cAMP in the maturing spermatozoa. By genetic manipulation of cAMP levels in developing spermatids and spermatozoa, the role of this second messenger in the acquisition of motility will be determined in situ. These studies will improve our understanding of the mechanisms controlling sperm maturation and will provide important information for a pharmacological improvement of sperm quality.